In order to investigate the function of nonmuscle myosin II-A, we created a nonmuscle myosin heavy chain II-A (NMHC II-A) null mouse by inserting the neomycin resistance cassette into the gene at exon 3. Previously, we have shown that ablation of nonmuscle myosin heavy chain II-B in mice results in abnormalities in the development of the heart and brain with the lethality occurring between E12 and the day of birth. The other organs of the B-/- mice appear normal, suggesting that NMHC II-A could substitute for II-B in these organs. In contrast to the NMHC II-B knockout, ablation of the NMHC II-A resulted in lethality at E7.5 or earlier, suggesting a role for NMHC II-A during early embryogenesis. NMHC II-A null embryos are smaller than normal littermates and are severely disorganized. The smaller size does not appear to be due to increased apoptosis, raising the possibility that the small size may be due to decreased cell proliferation. Immunocytochemistry of wild type mice at E7.5 showed the presence of NMHC II-A in all cell layers. At this stage, however, NMHC II-B was absent from the visceral endoderm. NMHC II-A null mice have a severely disorganized visceral endoderm suggesting that the absence of both isoforms of NMHC II contributed to the defect. Embryonic stem (ES) cells express both NMHC II-A and II-B. NMHC II-A -/- ES cells were generated by re-electroporation of NMHC II-A +/- cells followed by selection at increased concentrations of G418. Embryoid bodies, which mimic the early stages of development, have been made and assayed for the presence of markers of embryonic cell layers. Although some markers of visceral endoderm specification are present in the NMHC II-A null, heterozygous and wild type embryoid bodies, a number of the markers of visceral endoderm and mesoderm development are missing or diminished in the null embryoid bodies.